Disposition of metformin: Variability due to polymorphisms
of organic cation transporters OLIVER ZOLK Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-University of Erlangen-Nuremberg, Fahrstr. 17, 91054 Erlangen, Germany Abstract Considerable interindividual variability in clinical efcacy is recognized in the treatment of type 2 diabetes mellitus with the biguanide metformin. Metformin is a substrate of organic cation transporters, which play important roles in gastroin- testinal absorption, renal and biliary elimination, and distribution to target sites of substrate drugs. This raises the question of whether genetic variations in these transporters affect efcacy and risk of adverse events associated with metformin use. In this review, the pharmacogenetics of metformin is discussed in the light of the most recent literature. Overall, results from healthy volunteers support the notion that metformin pharmacokinetics can be affected by polymorphisms in genes encoding organic cation transporters. When considering the glycemic response to metformin in patients, however, the likely multifactorial nature of metformin response masks the effects of transporter polymorphisms observed in some clinical studies. Key words: Diabetes mellitus type 2 , genetic polymorphism , individualized medicine , metformin , organic cation transport proteins , pharmacogenetics , SLC22A1 transporter , SLC22A2 transporter , SLC47A1 transporter , SLC47A2 transporter Introduction Metformin is the principal biguanide drug that is used worldwide as an antihyperglycemic agent in patients with type 2 diabetes mellitus. In addition to type 2 diabetes, metformin is considered a therapeu- tic option for other diseases associated with insulin resistance, such as polycystic ovary syndrome (PCOS) and gestational diabetes. Metformin is already being prescribed to patients with PCOS, although it has not been approved for this indication (1). Its use in ges- tational diabetes is the subject of current clinical studies (2). A major action of metformin is suppression of hepatic glucose production. Although it has been used in the clinic since 1957, the direct molecular target of metformin remains unknown. However, there has been considerable progress in dening its pharmacological effects. Inhibition of hepatocyte glucose production by metformin is mediated by activation of the enzyme adenosine monophosphate- activated protein kinase (AMPK), a master sensor, integrator, and regulator of cellular and body energy homeostasis (3). AMPK activation is also implicated as a mechanism for the induction of skeletal muscle glucose uptake. One potential pathway by which metformin activates AMPK involves the upstream serine-threonine kinase 11 (STK11, also known as LKB1) (4). A recent report provides evidence that metformin is able to inhibit hepatic gluconeogenesis also in an LKB1- and AMPK-independent manner via a decrease in hepatic energy state (5). In addition to its efcacy in lowering glucose levels, metformin has the clinical advantages of inducing mild weight reduction and only a minimal risk of hypoglycemia. The United Kingdom Prospec- tive Diabetes Study in 1998 and subsequent studies and meta-analyses on the effects of metformin on outcomes conrm that metformin is one of the main Correspondence: Oliver Zolk, Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-University of Erlangen-Nuremberg, Fahrstr. 17, 91054 Erlangen, Germany. Fax: 49 9131 852277. E-mail: zolk@pharmakologie.uni-erlangen.de (Received 13 August 2010 ; accepted 14 December 2010 ) Annals of Medicine, 2011; Early Online, 111 ISSN 0785-3890 print/ISSN 1365-2060 online 2011 Informa UK, Ltd. DOI: 10.3109/07853890.2010.549144 A n n
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o n l y . 2 O. Zolk therapeutic options in type 2 diabetes mellitus, par- ticularly in overweight or obese patients, because it may prevent some macrovascular and microvascular complications and mortality (6 8). This favorable action prole has made metformin one of the most widely prescribed antidiabetic drugs worldwide. In the US market, metformin recently became one of the top ten prescribed products; more than 52 mil- lion prescriptions for its generic formulations were lled in 2009 (9). Metformin has some side-effects, principally gas- trointestinal, which occur in 20% 30% of patients and require discontinuation of the drug in less than 5% of patients (10). Very rarely, metformin causes lactic acidosis. The frequency of this side-effect has been estimated to be 4 5 cases per 100,000 patient years (11,12). In cases where metformin was impli- cated as the cause of lactic acidosis, metformin plasma levels of 5 g/mL were generally found (13), which is 4 5-fold higher than the maximum therapeutic steady-state plasma concentration (14). This suggests that metformin toxicity is associated with its accumulation in the body. The antidiabetic response to metformin varies considerably from patient to patient. Based on clinical trial experience, metformin reduces HbA 1C values by 0.8% to 3% (15). In patients using metformin monotherapy as their rst-ever antihyperglycemic agent, less than two-thirds of patients achieve a desired fasting glucose level or the HbA 1C goal of 7% (16,17). In routine clinical practice, the non- response rate to metformin in terms of achieving an HbA 1C of 7% within 1 year may be upwards of 50% (18). This incomplete response rate coupled with the waning effectiveness of metformin over time that occurs with most oral antidiabetic drugs (known as secondary failure due to the loss in insulin secretory capacity) highlights the need for personalized interventions to maintain tight glycemic control. Role of organic cation transporters in metformin pharmacokinetics It has been postulated that the observed variability in metformin response and the occurrence of lactic acidosis may be partially explained by variability in the pharmacokinetic disposition of metformin (19). The bioavailability of metformin is not complete, and large interindividual differences in bioavail- ability after oral administration in the range of 20% 70% have been described (20 22). Because metformin is not metabolized, this broad range probably reects differences in absorption rather than rst-pass metabolism. Furthermore, an inverse relationship between an orally administered dose of metformin and its bioavailability was observed (20,22), suggesting the involvement of a carrier- mediated saturable absorption process (23,24). Metformin is rapidly distributed following absorp- tion but does not bind to plasma proteins. Its vol- ume of distribution is large and can exceed 250 L, with the intestines, kidneys, and liver being the major organs of distribution (25,26). The clearance of metformin is primarily dependent on the single pathway of renal elimination, as metformin does not undergo relevant hepatic metabolism or biliary excretion. Because the renal clearance of metformin is much higher than the glomerular ltration rate, active tubular secretion by the kidney is the princi- pal mechanism of metformin elimination. Accord- ingly, renal metformin clearance correlates with excretory kidney function (i.e. creatinine clearance rate) (20,27). Data suggest that substantial variation in metformin renal clearance exists, and genetic fac- tors were found to contribute highly, by more than 90%, to the interindividual variation (28,29). Taken together, the pharmacokinetic characteristics of metformin and the inherited differences in met- formin renal elimination suggest that active trans- port processes are critical to the disposition of metformin. Because metformin is positively charged at phys- iological pH, it is not expected to diffuse freely Key messages Considerable interindividual differences in pharmacokinetics and clinical efcacy of metformin are recognized. Experimental studies in transgenic mice suggest that organic cation transporters are important determinants of metformin dis- position and pharmacologic action. Several non-synonymous SNPs in the human genes encoding organic cation trans- porters were identied and have been found to cause a loss-of-function phenotype of the affected transporter in vitro . Clinical studies suggest that polymorphisms in organic cation transporter genes, such as the p.270A S variant in the organic cation transporter 2 ( SLC22A2 ) gene, may affect renal clearance of metformin at least in homozygous carriers of the reduced-func- tion allele. An association of dened polymorphisms in organic cation transporters with glycemic response to metformin in patients has not yet been clearly established. A n n
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o n l y . Metformin disposition: role of transporter polymorphisms 3 through cell membranes but to cross biological membranes via carrier proteins. Indeed, metformin is a substrate for organic cation transporters (OCTs) in both the kidney and the liver (Figure 1). In humans, OCT1 (gene name SLC22A1 ) is expressed in the basolateral membrane of hepatocytes and is the primary mediator of hepatic metformin uptake (26,30,31). In contrast, OCT2 (gene name SLC22A2 ) is primarily expressed in the kidney and mediates uptake of metformin into the proximal tubule cells (31,32). In addition, metformin has been identied as a substrate for the multidrug and toxin extrusion (MATE) antiporters (33). MATE1 (gene name SLC47A1 ) is strongly expressed in the liver (canali- cular membrane), kidney (brush border membrane), and skeletal muscle (34). MATE2 (gene name SLC47A2 ) is predominantly expressed in the brush border membrane of renal proximal tubules (34). Thus, MATE transporters may contribute to renal excretion of metformin. The role of these transporters in the disposition of metformin was established in knock-out mouse models of Oct1 and Oct2. Oct1 (/) and Oct2 (/) mice are viable and display no obvious phenotypic abnormalities (35,36). However, Oct1 (/) mice show dramatically reduced hepatic uptake of tetra- ethylammonium (TEA; a prototypical organic cat- ion) and metformin (26,36,37), whereas renal excretion of metformin is virtually unchanged compared with wild-type mice (26). In mouse hepatocytes, deletion of Oct1 results in a reduction in the effects of metformin on AMPK phosphoryla- tion and gluconeogenesis. Thus, OCT1/Oct1 seems to be an important determinant of metformin action. When mice are given metformin, the blood lactate concentration signicantly increases in wild-type mice, whereas only a slight increase was observed in Oct1 (/) mice. Thus, Oct1 is respon- sible for the hepatic uptake of metformin, and the liver seems to be the key organ responsible for lactic acidosis (38). In Oct1/2 double knock-out mice, renal secretion of TEA is abolished, and plasma levels of TEA are substantially increased (35). Considering the differences in renal OCT expression between mice (Oct1 and Oct2) and human (OCT2), a combined deciency of Oct1 and Oct2 in mice is believed to better reect the effect of OCT2 deciency in humans. Thus, the latter study emphasizes the role of OCT2 in renal elimination of cationic drugs such as metformin. Targeted disruption of Mate1, which is expressed in renal proximal tubule cells where it mediates luminal secretion of organic cations into the urine, is expected to impair renal elimination of the cat- ionic drug metformin. Indeed, after intravenous administration of metformin, a 4-fold increase in the AUC of metformin was observed in Mate1 (/) mice compared to wild-type mice (39). The renal secretory clearance of metformin in Mate1 (/) mice was only 14% of that in Mate1 ( / ) mice. However, Mate1 (/) mice develop a nephropathy with creatinine clearance declining to only the half of that in wild-type mice (39). Although this obser- vation emphasizes the relevance of Mate1 to normal kidney function, the inherent kidney dysfunction observed in Mate1 (/) mice obscures the role of Mate1 in the renal clearance of metformin.
Figure 1. Localization and functional role of organic cation transporters in the pharmacokinetics of metformin. OCT organic cation transporter; MATE multidrug and toxin extrusion antiporter; PMAT plasma membrane monoamine transporter. Transporters in bold-face indicate major routes of metformin disposition in humans. A n n
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o n l y . 4 O. Zolk Implication of the polymorphic expression of OCTs in metformin pharmacokinetics and pharmacodynamics Organic cation transporter 1 (OCT1) Given the importance of OCT1 in metformin uptake into the liver and thus metformin activity, several pharmacogenetic studies have focused on polymor- phisms in the gene encoding OCT1, SLC22A1 , as modiers of the glycemic response. Human SLC22A1 is a highly polymorphic gene (Table I). Functional studies in cell-based models have established that among the naturally occurring protein variants, the p.61R C, p.189S L, p.220G V, p.401G S, p.420del, and p.465G R mutants reduce OCT1 function and thus reduce or eliminate metformin uptake (37). The decrease in uptake by some variant proteins is probably due to their cytosolic retention and reduced expression on the plasma membrane, as was shown for the SLC22A1 p.465G R and p.61R C variant proteins in transfected cells (37,40). Recently, Nies et al. conrmed these exper- imental data in a clinical study by demonstrating that the presence of the SLC22A1 c.262T C (p.61R C) SNP in patients strongly correlates with decreased liver OCT1 protein expression (41). Consistent with the previously described knock-out studies, phosphorylation of AMPK following metformin administration is reduced in cells expressing the non-functional or reduced-function variants, com- pared with cells expressing wild-type OCT1. Based on this experimental observation, it was proposed that OCT1 mediates the rst step in the response pathway to metformin and that genetic variation in SLC22A1 may modulate the response to metformin in humans. Shu et al. rst reported that individuals with any of the four reduced-function alleles (p.61R C, p.401G S, p.420del, or p.465G R) have a signi- cantly decreased glucose-lowering response to metformin compared with reference allele carriers (Table II summarizes results from clinical studies). To determine the glycemic response, study partici- pants (healthy volunteers) underwent oral glucose tolerance testing before and after two doses of met- formin. Individuals with the reference sequence demonstrated a 7% reduction in the area under the glucose concentration-time curve (glucose AUC), whereas the glucose-AUC in variant allele carriers increased from base-line by 8% (Figure 2). Shu et al. also investigated the effect of SLC22A1 variants on the pharmacokinetics of metformin in the same study cohort. Unlike prior animal studies, which demonstrated no difference in the pharmacokinetic prole in wild-type or Oct1 (/) mice (26), pharma- cokinetics differed across the SLC22A1 genotype groups in humans, with a signicantly higher met- formin AUC ( 19%), higher maximal plasma concen- tration (C max ; 15%), and lower oral volume of distribution (V/ F ; 47%) in individuals carrying a reduced-function allele. These effects may be at least partially explained by a much lower hepatic uptake of metformin in individuals with a variant allele. Of note, renal metformin clearance was unchanged across SLC22A1 genotypes in that study, whereas in a subsequent study Tzvetkov et al. noted an increase in renal metformin clearance in healthy volunteers carrying a SLC22A1 reduced-function allele (42). Studies in diabetic patients were also conducted to study pharmacodynamics. Zhou et al. investigated whether the two most common reduced-function SNPs in SLC22A1 , p.61R C and p.420del, decrease the glycemic response in patients with type 2 diabe- tes (43). In that study, a series of drug response mod- els for metformin were assessed, including short- and mid-term HbA 1C reduction, reaching the treatment target of HbA 1C of 7%, and time to monotherapy failure, in a large population-based study of 1,531 patients recruited to GoDARTS (Genetics of Diabe- tes Audit and Research Tayside). The study result was essentially negative, i.e. the SLC22A1 variants p.61R C and p.420del did not affect the initial HbA 1C reduction, the chance of achieving a treat- ment target, the average HbA 1C on monotherapy up to 42 months, or the hazard of monotherapy failure (43). In another study, Shikata et al. analyzed vari- ants of SLC22A1 and SLC22A2 in 33 Japanese dia- betic patients with variable treatment efcacy to metformin (44). None of the identied polymor- phisms in either gene was associated with metformin responder status, which was dened by an absolute reduction of 0.5% in HbA 1C within the rst 3 months of metformin therapy. However, when clini- cal variables were included and multivariate statistics were applied, two SNPs in SLC22A1 , c.43T G and c.1222A G, were negative and positive predic- tors, respectively, for the metformin responder sta- tus. The biological basis of these associations remains somewhat elusive, as the c.1222A G SNP is not associated with altered metformin transport in cel- lular models, and expression studies in human liver tissue samples did not reveal any allelic differences in OCT1 expression. Becker et al. analyzed associa- tions between 11 tagging SNPs in SLC22A1 and changes in the HbA 1C level in a subcohort of 102 incident metformin users from the population-based Rotterdam study (45). No signicant associations were observed except for the intronic rs622342 A C SNP. For each minor C allele of this variant, the reduction in HbA 1C levels in diabetic patients was 0.28% less. However, the mechanism by which this SNP, which is not in linkage disequilibrium with A n n
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o n l y . Metformin disposition: role of transporter polymorphisms 5 known reduced-function SLC22A1 SNPs or associ- ated with renal metformin clearance (42), affects the glycemic response to metformin remains unclear. Similar to patients with type 2 diabetes, great variability in the clinical response to metformin has also been observed in women with PCOS. To test whether SNPs in SLC22A1 contribute to the variability in treatment response, Gambineri et al. conducted a prospective study in 150 patients of European descent with PCOS who were treated with metformin for 6 months (46). Carriers of at least one of the four SLC22A1 reduced-function alleles Table I. Polymorphisms investigated in clinical studies, their impact on the in-vitr o transport function, and their allele frequency in different ethnic populations. Nucleotide change Amino acid change Activity (37,47,62) European (ethnic subpopulation, number of tested individuals) (40,41) African (ethnic subpopulation, number of tested individuals) (40) Asian (ethnic subpopulation, number of tested individuals) (40,47,63) SLC22A1 (OCT1) c.262T C p.61R C 0.10 (EC 150) 0.07 (EA 200) 0 (AA 200) 0 (AS 60) 0 (JA 116) c.289C A p.97Q K 0.02 (JA/HC 59) c.350C T p.117P L 0.02 (JA 66) c.616C T p.206R C 0.01 (JA 66) c.659G T p.220G V 0 (EA 200) 0.01 (AA 200) 0 (AS 60) c.848C T p.283P L c.859C G p.287R G c.1201G A p.401G S 0.01 (EC 150) 0.01 (EA 200) 0.01 (AA 200) 0 (AS 60) 0 (JA 116) c.1222A G p.408M V 0.60 (EA 200) 0.74 (AA 200) 0.76 (AS 60) c.1256delATG p.420del 0.17 (EC 150) 0.19 (EA 200) 0.03 (AA 200) 0 (AS 60) 0 (JA 116) c.1393G A p.465G R 0.04 (EC 150) 0.04 (EA 200) 0 (AA 200) 0 (AS 60) 0 (JA 116) Nucleotide change Amino acid change Activity (50 52) European (50,51) African (50) Asian (50,51,64,65) SLC22A2 (OCT2) c.495G A p.165M I 0 (EA 200) 0.01 (AA 200) 0 (AS 60) c.596C T p.199T I 0 (EC 100) 0 (HC 100) 0.01 (KA 150) 0 (VA 100) c.602C T p.201T M 0 (EC 100) 0 (HC 100) 0.01 (KA 150) 0.02 (VA 100) c.808G T p.270A S ( ) 0.16 (EA 200) 0.11 (AA 200) 0.13 (HC 400) 0.14 (HC 100) 0.17 (JA 116) 0.09 (AS 60) c.1198C T p.400R C 0 (EA 200) 0.02 (AA 200) 0 (JA 116) 0 (AS 60) c.1294A C p.432K Q 0 (EA 200) 0.01 (AA 200) 0 (JA 116) 0 (AS 60) Nucleotide change Amino acid change Activity (54,55) European (54,55) African (54,55) Asian (54,55) SLC47A1 (MATE1) g.66T C / (promoter) 0.32 (EA 68) 0.45 (AA 68) 0.23 (CA 68) c.404T C p.159T M 0 (EC 253) 0 (AA 95) 0 (TA 95) 0.01 (JA 95) c.1012G A p.338V I 0 (EC 253) 0.05 (AA 95) 0.10 (TA 95) 0.01 (JA 95) AA African American; AS Asian American; CA Chinese American; EA European American; EC European Caucasian; HC Han Chinese; JA Japanese Asian; KA Korean Asian; TA Tanzanian African; VA Vietnamese Asian; reduced transport function; increased transport function; no change. A n n
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o n l y . 6 O. Zolk (p.61R C, p.401G S, p.420del, or p.465G R) had a signicantly decreased cholesterol- and triglycer- ide-lowering response to metformin compared with reference allele carriers. Glucose tolerance testing at base-line and at the end of the treatment revealed that the SLC22A1 genotype is a determinant of the insulin response to metformin, but not of the glucose AUC. Given that the lipid-lowering effect is one Table II. Published pharmacogenetic studies investigating associations between polymorphisms in cation transporter genes and transporter expression, metformin pharmacokinetics, or glycemic effects of metformin therapy. Study population Outcome (versus reference) Healthy volunteers: carriers of at least one reduced-function SLC22A1 allele (p.61R C, p.401G S, p.420del, or p.465G R) ( n 12) versusreference allele carriers ( n 8) Metformin kinetics (at least one reduced-function allele versus ref.): AUC ( 19%) C max ( 15%) CL/ F ( 38%) V/ F ( 47%) CL R
(66) Healthy male volunteers ( n 103): association of SNPs in SLC22A1 , SLC22A2 , SLC22A4 , and SLC47A1 with metformin renal clearance Presence of SLC22A1 reduced-function alleles was associated with increased renal metformin clearance (42) Healthy volunteers: SL22A2 p.270A S heterozygous ( n 5) and p.270A S homozygous ( n 4)versusreference allele carriers ( n 6) Metformin kinetics (808 TT versus ref.): AUC C max CL R ( 26%) In the presence of cimetidine, metformin CL was decreased in all participants, but the decrease was lower in TT than GG carriers (67) Healthy volunteers: SLC22A2 p.199T I heterozygous ( n 3), p.201T M heterozygous ( n 2), p.270A S heterozygous ( n 6), and p.270A S homozygous ( n 6)versus reference allele carriers ( n 9) Metformin kinetics (p.270 SS versus ref.): AUC ( 73%) C max ( 63%) CL/ F ( 32%) CL R ( 51%) In heterozygous allele carriers, only minor (p.270A S) or no (p.199T I , p.201T M) changes were seen (53) Diabetic patients: heterozygous carriers of variant SLC47A1 (p.64G D, p.125L F, p.328D A) and SLC47A2 (p.211G V) alleles ( n 7) versus reference allele carriers ( n 41) Metformin kinetics: CL/ F (57) Tissue samples from human livers; heterozygous carriers of the SLC22A1 p.61R C allele ( n 29) versus reference allele carriers ( n 118) SLC22A1 mRNA expression (41) Human kidney tissue samples from donors with at least one SLC47A1 g.66T C variant allele ( n 26) versus samples from donors with the reference genotype ( n 12) SLC47A1 mRNA expression ( 34%) (54) Healthy volunteers: carriers of at least one reduced-function SLC22A1 allele (p.61R C, p.401G S, p.420del, or p.465G R) ( n 12) versus reference allele carriers ( n 8) Oral glucose (75 g) tolerance test before and after two doses of metformin: Effect of metformin on glucose AUC (37) Women with PCOS: carriers of at least one reduced-function SLC22A1 allele (p.61R C, p.401G S, p.420del, or p.465G R) ( n 66) versus reference allele carrier ( n 84) Oral glucose (75 g) tolerance test: Effect of metformin on glucose AUC after 6 months of treatment Effect of metformin on insulin AUC after 6 months of treatment ( 2 variant alleles versus control) (46) Patients with diabetes mellitus ( n 1,531): SLC22A1 p.61R C and p.420del variant allele carriers versus reference allele carriers Variants did not affect the initial HbA 1C
reduction, the chance of achieving a treatment target, the average HbA 1C on monotherapy up to 42 months, or the hazard of monotherapy failure (43) Diabetic patients: responders to metformin ( n 24) versus non-responders ( n 9); correlation of SLC22A1 and SLC22A2 polymorphisms (identied by screening the respective genes) with response to metformin c.43T G and c.1222A G were negative and positive predictors, respectively, for the efcacy of metformin (OCT1 mRNA levels tended to be lower in human livers with the c.1222A G variant allele) (44) Diabetic patients: association of SNPs in SLC47A1 with reduction in HbA 1C level after initiation of metformin treatment in a population-based cohort ( n 116) Among genotypes, the intronic SNP rs2289669 (c.922158G A) was signicantly associated with an increased glucose-lowering effect (56) Diabetic patients: association of SNPs in SLC22A1 with reduction in HbA 1C level after initiation of metformin treatment in a population-based cohort ( n 102) Among genotypes, the SNP rs622342 was signicantly associated with an increased glucose-lowering effect (45) A n n
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o n l y . Metformin disposition: role of transporter polymorphisms 7 aspect of the hepatic action of metformin and that OCT1 plays a trigger role in hepatic metformin uptake, this observation is in agreement with the hypothesis that polymorphisms in SLC22A1 contribute to the variable response to metformin. The genotypic frequencies of the non-synonymous, reduced-function polymorphisms in SLC22A1 vary substantially among different races or ethnicities. In general, they are rare in Asian populations and most frequent in Caucasians (Table I). Although this observation may be biased because genetic variants in SLC22A1 have been investigated and identied largely in European populations, a recent screen for genetic variants in SLC22A1 in Chinese and Japa- nese populations did not substantially change the conclusion (47). Given the important role of OCT1 in hepatic uptake and action of metformin, inter- ethnic differences in the frequency of SLC22A1 reduced-function SNPs may be associated with inter-ethnic differences in the pharmacodynamic prole of metformin (48). Signicant heterogeneity in metformin efcacy by ethnic group, however, was not observed (49). Organic cation transporter 2 (OCT2) Given the importance of OCT2 in metformin phar- macokinetic disposition, some pharmacogenetic studies have focused on polymorphisms in the gene encoding OCT2, SLC22A2 , as a modier of met- formin renal clearance. To date, almost 500 variable sites in SLC22A2 have been identied. Thirteen cause non-synonymous amino acid changes, and most are present at frequencies of less than 1%. Some variants such as p.165M I, p.199T I, p.201T M, and p.400R C lead to clearly reduced activity compared to the OCT2 reference (50,51), whereas the p.270A S (c.808G T) variant has more subtle effects on transporter function in vitro (52). Most pharmacogenetic studies performed in healthy volunteers have focused on the last-men- tioned SNP because it is the only common coding polymorphism in SLC22A2 , with an allele frequency of about 15% regardless of the ethnic background. Overall, these studies suggest that the SLC22A2 c.808G T SNP has no signicant impact on renal metformin clearance in heterozygous carriers (Fig- ure 3). A marked reduction in metformin clearance by almost 40%, however, was observed in volunteers with the homozygous variant TT genotype. For this polymorphism, the results obtained so far (Figure 3) t well with a recessive model of genotype pheno- type interaction, i.e. changes in renal metformin clearance occur only when both c.808 alleles are dys- functional. Thus, it is possible that patients with the SLC22A2 c.808 GG or GT genotype will require a different dosage of metformin to achieve optimal glucose control compared with homozygous TT allele carriers. However, data are lacking to show whether SLC22A2 c.808G T genotype-dependent changes in metformin pharmacokinetics (demon- strated only in healthy volunteers so far) translate into changes in metformin glycemic response in patients. Song et al. identied few heterozygous carriers of the rare SLC22A2 c.596C T (p.199T I) and c.602C T (p.201T M) variants (which exhibit reduced transport of metformin in cellular assays) and compared metformin clearance in these individuals with that in wild-type allele carriers (53). These stud- ies suggest that the presence of one reduced-function SLC22A2 c.596C T or c.602C T allele leads to a reduction in renal metformin clearance comparable to that observed in homozygous SLC22A2 c.808 TT carriers. The small number of tested individuals, how- ever, precludes denite conclusions. Because these SNPs occur only in some ethnic subpopulations and at a frequency of 1%, their clinical impact in view of a population-based pharmacogenetic screening approach is rather limited. Multidrug and toxin extrusion antiporters (MATE1, MATE2) As described above, the MATE1 and MATE2 trans- porters, which are located in the canalicular mem- brane of hepatocytes and in the brush border of the
Figure 2. Association of SLC22A1 (OCT1) variants with response to metformin in healthy volunteers. Oral glucose (75 g) tolerance test was performed before (base-line) and after two doses of metformin. The glucose AUC was calculated from the time course of plasma glucose concentrations. Comparison of healthy individuals with only reference SLC22A1 alleles ( n 8) and those with at least one reduced-function allele in SLC22A1 : p.61R C, p.401G S, p.420del, or p.465G R ( n 12). Data are from (37). A n n
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o n l y . 8 O. Zolk renal epithelium, are responsible for the nal step of the excretion of cationic compounds into bile and urine, respectively. Metformin is a substrate of these transporters, and therefore these transporters may be involved in metformin excretion. Several poly- morphisms have been identied and characterized in the gene encoding MATE1, SLC47A1 . SLC47A1 mRNA expression, for example, is signicantly lower in human kidney samples from individuals who are homozygous or heterozygous for the SLC47A1 g.66T C SNP in the basal promoter in comparison with samples from homozygous refer- ence allele carriers (54). In-vitro experiments sug- gest that the reduced transcriptional activity of SLC47A1 g.66T C results from a reduction in the binding potency of the transcriptional activator, activating protein-1, and an enhanced binding potency of the repressor, activating protein-2 repres- sor, to the mutant basal promoter region (54). In cellular models, two non-synonymous SNPs in SLC47A1 , p.159T M (c.404T C) and p.338V A (c.1012G A), cause a signicant loss in transporter activity for metformin (55). Unfortunately, no stud- ies have been reported that examine the effect of the experimentally characterized promoter or reduced- function variants on the pharmacokinetics and pharmacodynamics of metformin in patients. Based on data from the population-based Rot- terdam cohort study, a signicant association between the tagging rs2289669 G A SNP in SLC47A1 and metformin response in a study sample of 116 inci- dent metformin users was observed (56). The SNP was associated with an increased glucose-lowering effect. For each minor A allele, the HbA 1C reduction was 0.3% larger (56). However, the small sample size, the limited clinical phenotype, and the lack of a rep- lication study limit the informative value of this study. In another clinical study, Toyama et al. observed no differences in metformin disposition (evaluated by recording the plasma concentration time prole after oral administration of metformin) between diabetic patients carrying a heterozygous variation in SLC47A1 or SLC47A2 and patients with the reference genotype (57). Similarly, no differences were observed in met- formin pharmacokinetics between heterozygous Mate1 ( /) knock-out mice and Mate1 ( / ) wild- type mice, whereas metformin kinetics were mark- edly affected in homozygous Mate1 (/) knock-out mice. Thus, the authors hypothesized that heterozy- gous variants in SLC47A1 and SLC47A2 do not sub- stantially contribute to the interindividual variation in metformin pharmacokinetics (57). The postulated recessive model of the SLC47A1/2 genotype pheno- type interaction seems to be plausible particularly because two MATE efux transporters with substrate redundancies are expressed, such that one MATE transporter can compensate at least partially for an inherently reduced expression or function of another MATE transporter. Conclusion Several non-synonymous, promoter and deletion- type variants in the SLC22A1 , SLC22A2 , and
Figure 3. Effects of polymorphisms in genes encoding the organic cation transporter 2 (OCT2, SLC22A2 ) on renal clearance (CL R ) of metformin in healthy volunteers. Synopsis/meta-analysis (analysis method: xed effect inverse variance model) of published studies (42,53,67,68). Modied from (69). A n n
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o n l y . Metformin disposition: role of transporter polymorphisms 9 SLC47A1 organic transporter genes exhibit reduced transporter activity of metformin in experimental studies. The best-studied SNP in humans with respect to the pharmacokinetics of metformin is the SLC22A2 p.270A S SNP, which shows a recessive mode of genotype phenotype interaction with an almost 40% reduction in renal metformin clearance in homozygous carriers of the minor allele. At the level of pharmacokinetics in healthy individuals, the results are less controversial; however, the results are inconsistent when focusing on the glycemic response to metformin in patients with type 2 diabetes. In clinical studies, metformin has a decreased effect on glucose tolerance in healthy individuals who carry reduced-function polymorphisms of SLC22A1 . However, these ndings were not conrmed in a ret- rospective study of 1,531 metformin-treated diabetic patients, which showed no differential effect in HbA 1C reduction when considering the two most frequent index variants at this locus. Why have we so far failed to establish genotypes of organic cation transporter genes ( SLC22A1 , SLC22A2 , SLC47A1 , SLC47A2 ) as validated predic- tors of pharmacokinetics and clinical response to metformin in patients? Possibly, the effects of single genetic polymorphisms in transporter genes are too small against a noisy background caused by environmental factors, age, gender, and co-morbid- ities. In fact, in a study of young female patients with PCOS who presumably had few co-morbidities (i.e. with more tight control of non-genetic covari- ates), the SLC22A1 genotype was found to be a sig- nicant determinant of lipid and insulin responses to metformin (46). Estimates of the contribution of genes and envi- ronment to the variation in renal metformin clear- ance suggest a strong genetic inuence in Caucasian and Asian populations (genetic component r GC 0.9) (28,29). However, caution is needed in interpreta- tion of these data obtained from two small popula- tions of young healthy subjects. Both studies clearly failed to reach satisfactory statistical power (58). Moreover, the value of heritability applies only to the population in which it was established. Assum- ing a given extent of genetic control, the heritability of renal clearance of metformin tends to be larger the more uniform the tested population. Although the exact value is not known, an r GC value 0.9 certainly under-estimates the environmental com- ponent of renal metformin clearance in a general population, as indicated by calculations of Tzvetkov et al. Considering only the two non-genetic param- eters kidney function and age, these parameters are explaining 42% and 9% of the variation in renal clearance of metformin, respectively (42). Three decades ago, Sirtori et al. and Tucker et al. observed that renal metformin clearance is highly correlated with creatinine clearance (20,27). Cholestasis was also identied as an important non-genetic factor that is associated with markedly reduced OCT mRNA and protein expression in the human liver (41), potentially affecting hepatic disposition of metformin. Moreover, variations in genes other than those encoding OCTs and MATEs are likely to modulate the pharmacokinetics or response to metformin ther- apy. For example, some recent studies suggest a role for STK11 , a molecular target gene of metformin, in the treatment response to metformin (59,60). Another candidate is SLC29A4 , which encodes the plasma membrane monoamine transporter (PMAT). PMAT transports metformin, is expressed in human intestine, and may play a role in the intestinal absorp- tion of metformin (61). Unlike warfarin in which 40% of the variation in treatment response is due to two genes, VCORCI and CYP2C9 , the pharmacoge- netics of metformin seems to not be one of the few cases in which one or a few genes determines a large proportion of the variation of the response to a drug. One reason for the polygenic phenotype of met- formin pharmacokinetics may be the functional redundancy of some human transporters. Metformin is a substrate of human MATE1 and MATE2 trans- porters, which are expressed at the renal brush bor- der membrane. Another example is OCT1 and OCT3, which are localized at the basolateral hepa- tocyte membrane (41). Although OCT3 is expressed in the human liver at lower levels than OCT1, OCT3 transports metformin with a higher efcacy than OCT1 and thus may counterbalance impaired OCT1 function (41). In conclusion, investigation of polymorphisms in organic cation transporters and attempts to link the variants with the pharmacokinetics and phar- macodynamics of metformin have provided valu- able information about the role of these transporters in the disposition of metformin in humans. Knowing the biology of the polymor- phisms in genes dening the disposition of met- formin (a prototypic cationic drug) may bring us a step closer to clinical application in terms of individualized drug therapy. Acknowledgements I thank MF Fromm for critical reading of the manuscript. Declaration of interest: The author states no conict of interest and has received no payment in preparation of this manuscript. A n n
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